Ubiquitin ligase KPC1 promotes processing of P105 NF-κB1 to p50, eliciting strong tumor suppression
The invention provides a method for treating cancer comprising the step of administering a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 or an agent which up-regulates KPC1. In some embodiments, there is also provided a method for treating cancer comprising the step of administering a therapeutically effective amount of p50, a peptide which is at least about 70% homologous to the p50 or an agent which up-regulates p50.
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This application claims the benefit of U.S. Provisional Application No. 62/104,883, filed on Jan. 19, 2015 and entitled UBIQUITIN LIGASE KPC1 PROMOTES PROCESSING OF P105 NF-KAPPAB1 TO P50, ELICITING STRONG TUMOR SUPPRESSION, which is incorporated in their entirety herein by reference.
BACKGROUND OF THE INVENTIONThe NF-κB family of transcription factors is involved in regulation of a variety of genes that control the immune and inflammatory response, cell survival and death, proliferation and differentiation. Recently—150 years after Rudolf Virchow discovered the infiltration of tumors with leukocytes and proposed a linkage between chronic inflammation and malignant transformation—it has been shown that the mechanism(s) that underlies this linkage is mediated largely by the NF-κB family of transcription factors (Ben-Neriah and Karin, 2011; DiDonato et al., 2012). NF-κB is overexpressed in numerous tumors. It up-regulates expression of anti-apoptotic genes such as IAPs, cell cycle promoters, and growth factors and their receptors (DiDonato et al., 2012). Nevertheless, in some cases NF-κB was shown to display strong tumor suppressive characteristics (Perkins, 2012; Pikarsky and Ben-Neriah, 2006). For example, it is involved in regulation of activation-induced apoptosis of T lymphocytes (Ivanov et al., 1997), and in inducing cell cycle arrest and cell death caused by repression of Bcl2, XIAP, Bcl-XL, Cyclin D1 and c-Myc that occurs after cell damage. The arrest and death are mediated by p52 dimers (Barre et al., 2010; Barre and Perkins, 2007). Also, it was shown that NF-κB1−/− cells accumulate alkylator-induced mutations, and NF-κB1−/− mice develop more lymphomas following alkylating agent-induced DNA damage, again suggesting that NF-κB1 can act as a tumor suppressor (Voce et al., 2014).
The family members are mostly heterodimers where one of the subunits—p52 or p50, is the product of limited, ubiquitin- and proteasome-mediated processing of a longer (and inactive) precursor, p100 or p105, respectively (Betts and Nabel, 1996; Fan and Maniatis, 1991). The other subunit is typically a member of the Rel family of proteins (RelA—p65, RelB or c-Rel). At times, p50 and p52 can generate homodimers that cannot act as transcriptional activators since they lack a transactivation domain present in the Rel proteins. In unstimulated cells, the NF-κB dimers are sequestered in the cytosol attached to ankyrin repeats (ARs) of IκB inhibitory proteins (IκB, Bcl3, p100 and p105). A broad array of extracellular signals stimulate degradation of the IκB proteins, resulting in translocation of the dimers to the nucleus where they initiate different transcriptional programs (Rahman and McFadden, 2011).
Proteasomal processing of p105 occurs under both basal conditions and following stimulation, and requires prior ubiquitination (Cohen et al., 2004; MacKichan et al., 1996). One element that was shown to be important in the processing is a long Gly-Ala repeat in the middle of p105 that may serve as a proteasomal “stop signal” (Lin and Ghosh, 1996). In addition to processing, p105 can also undergo complete degradation, releasing NF-κB dimers anchored to its C-terminal ARs domain. Following stimulation, p105 is phosphorylated on serine residues 927 and 932 by IκB kinase (IKKβ) (Salmeron et al., 2001). This modification recruits the beta-Transducin Repeat Containing Protein βTrCP) E3 (Orian et al., 2000), resulting in complete degradation of the molecule (Heissmeyer et al., 2001). The ligase(s) involved in processing of p105 under basal conditions as well as following stimulation has remained elusive.
SUMMARY OF THE INVENTIONThe invention is directed to a method for treating cancer comprising the step of administering a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1; a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1; or a complex comprising KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent that upregulates KPC1; or the fused protein that comprises KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, to a subject in need, thereby treating cancer.
According to another embodiment, the method for treating cancer comprising the step of administering a therapeutically effective amount of functionally related variant of the KPC1 or a functionally active fragment of KPC1, a fused protein comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1 or a complex comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1, or the fused protein that comprises the functionally related variant of the KPC1 or the functionally active fragment of KPC1, to a subject in need, thereby treating cancer.
According to another embodiment, the method for treating cancer comprising the step of administering a therapeutically effective amount of p50, peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or an agent which upregulates p50; a fused protein comprising p50 or the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which up-regulates p50; or a complex comprising p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50 or the fused protein that comprises p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50, to a subject in need, thereby treating cancer.
According to another embodiment, the method for treating cancer comprising the step of administering a therapeutically effective amount of functionally related variant of the p50 or a functionally active fragment of p50, a fused protein comprising the functionally related variant of the p50 or the functionally active fragment of p50 or a complex comprising either the functionally related variant of the p50 or the functionally active fragment of p50, the fused protein that comprises the functionally related variant of the p50 or the functionally active fragment of p50 to a subject in need, thereby treating cancer.
According to another embodiment, the method of treating cancer by administering a therapeutically effective amount of a nucleic acid sequence that encodes to KPC1 or to a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or a nucleic acid that encodes to a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or a nucleic acid that encodes to an agent which up-regulates KPC1 or to a fusion protein comprising thereof.
According to another aspect, the present invention provides a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4.
According to another embodiment the KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 is attached to a heterologous amino acid sequence.
According to another embodiment the heterologous amino acid sequence comprises an immunoglobulin amino acid sequence.
According to another embodiment the immunoglobulin amino acid sequence comprises IgG.
According to another aspect, the present invention provides a nucleic acid encoding a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4.
According to another aspect, the present invention provides a vector comprising a nucleic acid encoding a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4.
According to another aspect, the present invention provides a cell transformed with a vector comprising a nucleic acid encoding a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4.
According to another aspect, the present invention provides a complex comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 and non-proteinaceous or proteinaceous moiety.
According to another embodiment the non-proteinaceous moiety is polyethylene glycol (PEG) or derivative thereof, polyvinyl pyrrolidone (PVP), divinyl ether, albumin, maleic anhydride copolymer (DIVEMA), polysialic acid (PSA), poly(styrene comaleic anhydride) (SMA), hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), glyme or polyisopropylacrylamide or any combination thereof.
According to another aspect, the present invention provides a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1; a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1; or a complex comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent that upregulates KPC1, or the fused protein that comprises KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, for treating a cancer.
According to another aspect, the present invention provides a therapeutically effective amount of functionally related variant of the KPC1 or a functionally active fragment of KPC1, a fused protein comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1 or a complex comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1, or the fused protein that comprises the functionally related variant of the KPC1 or the functionally active fragment of KPC1 for treating cancer.
According to another aspect, the present invention provides a therapeutically effective amount of p50, peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or an agent which upregulates p50; a fused protein comprising p50 or the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which up-regulates p50; or a complex comprising p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50 or the fused protein that comprises p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50 for treating cancer.
According to another aspect, the present invention provides a therapeutically effective amount of functionally related variant of the p50 or a functionally active fragment of p50, a fused protein comprising the functionally related variant of the p50 or the functionally active fragment of p50 or a complex comprising either the functionally related variant of the p50 or the functionally active fragment of p50, the fused protein that comprises the functionally related variant of the p50 or the functionally active fragment of p50 for treating cancer.
According to another aspect, the present invention provides a therapeutically effective amount of a nucleic acid sequence that encodes to KPC1 or to a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or a nucleic acid that encodes to a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or a nucleic acid that encodes to an agent which up-regulates KPC1 or to a fusion protein comprising thereof for treating a cancer.
According to another aspect, the present invention provides a method for producing p50 comprising contacting a cell culture preparation which expresses p105 with KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1, thereby producing p50.
In some embodiments of the invention the cancer to be treated by the methods and active ingredients of the invention is breast cancer, bone osteosarcoma or glioblastoma.
In some embodiments of the invention the agent used in the method of treating cancer which upregulates KPC1 activity or expression increases the level of KPC1 or increases the binding between KPC1 and p105.
In some embodiments of the invention, the agent which upregulates KPC1 is an agonist to KPC1 which may be a chemical agent or a small molecule.
The invention will now be described in connection to certain Examples and embodiments, in a non-limiting manner, with reference to the following illustrative figures, so that it can be more fully understood. In the drawings:
Below at the lower panel is a peptide coverage map of mouse KPC2. The peptides were identified through mass spectrometric analysis of the E3-containing fractions resolved by the last, Heparin-based column. Residues marked in Bold and Italics denote differences in sequence between mouse and rabbit.
24 hr after transfection (in the experiments depicted under Panels C and D), cycloheximide was added for the indicated times, and cells were lysed, resolved via SDS-PAGE, and proteins visualized using anti-FLAG, anti-KPC1 or anti-actin. Processing and degradation were assessed as described under
Chx denotes cycloheximide. Actin was used to ascertain equal protein loading.
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Suppression of colony formation by overexpressed KPC1 or p50 in MDA-MB 231 (
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All scale bars, 100 m. Tumors were grown in mice and stained as described under Experimental Procedures.
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Validation of the specificity of the anti-KPC1 antibody. Immunohistochemistry of KPC1 in HEK293 cells that were transfected with control siRNA (left), siRNA to silence KPC1 (middle) or with Myc-KPC1 (right). All scale bars, 20 μm.
and (C) Validation of the specificity of the anti-p50 antibody. Upper panel: Immunohistochemistry of p50 in xenografts of U87-MG cells stably expressing V0, Myc-KPC1, FLAG-p50 or shRNA to p105. Lower panel: immunohistochemistry of p50 in xenografts of U87-MG cells stably expressing Myc-KPC1 in the presence or absence of a specific blocking peptide (10 μg/ml) to the anti-p50 antibody. All scale bars, 100 μm.
DETAILED DESCRIPTION OF THE PRESENT INVENTIONIn the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.
In an embodiment of the invention, NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. In an embodiment of the invention, KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. In an embodiment of the invention, overexpression of KPC1 promotes inhibition of tumor growth mediated probably via excessive generation of p50. In an embodiment of the invention, excessive p50 down regulates p65, explaining the possible lack of the ‘canonical’, p50∩p65 tumor promoting NF-κB. Transcript analysis reveals increased expression of genes associated with tumor suppressive signals. Overall, in an embodiment of the invention, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.
In an embodiment of the invention, KIP1 ubiquitination-promoting complex (KPC) was identified as the Ub ligase that is involved in both basal and signal-induced processing of p105. KPC is a heterodimer made of KPC1 (RNF123) and KPC2 (UBAC1). It was shown to degrade the cyclin-dependent kinase inhibitor p27KIP1 in the G1 phase of the cell cycle (Kamura et al., 2004). In an embodiment of the invention, KPC1 is a RING-finger protein that serves as the ligase. In an embodiment of the invention, KPC2 interacts with ubiquitinated proteins and with the proteasome via its two Ub-associated domains and a Ub-like domain, acting as a shuttle that promotes the degradation of p27KIP1. It was also shown to stabilize KPC1 (Hara et al., 2005).
The vast majority of substrates of the Ub proteasome system are completely degraded. One intriguing and exceptional case is that of the p105 precursor of NF-κB that can be either completely degraded or processed in a limited manner to yield the p50 active subunit of the transcription factor. The “decision-making” mechanism resulting in one of the two distinct processes has remained largely elusive. The βTrCP Ub ligase has been identified as the tagging enzyme involved in complete degradation of p105, whereas the ligase involved in processing has remained unknown. The KPC complex has been identified as the putative p105-processing ligase (
Now that the two E3s involved in degradation and processing of p105 have been identified, it is still not known why ubiquitination by one enzyme results in a completely different fate of p105 than ubiquitination by the other, and what determines the timing of the two reactions. It is possible that the two ligases catalyze the formation of chains that differ in their anchoring sites, length and/or internal linkages. These in turn affect the recognition and mechanism of action of the 26S proteasome. As for timing, it can be that different physiological conditions and/or the degree of saturation of the ARs with bound p50s are involved in the “decision-making” process of whether the molecule will be processed or destroyed completely.
Studying the biological implications of manipulating KPC1 revealed that in one embodiment, suppresses anchorage-independent growth in a manner that is dependent on its ligase activity and the presence of p105. A corollary strong tumor suppressive effect was demonstrated in xenografts of human tumors (see in the Examples and
Indeed, following overexpression of KPC1 or p50, there is a decrease in the level of what is probably the ‘canonical’ tumorigenic NF-kB (p50⋅p65;
A strong correlation between the expression of KPC1 and that of p50 in human tumors has been found (
As shown in the Examples section, KPC1 promotes p105 NF-kB1 (SEQ ID No. 1) proteasomal processing to p50 (SEQ ID No. 3) which results in tumor suppression Further, overexpression of KPC1 (SEQ ID No. 4) or p50, suppresses tumor growth.
The amino acid sequence of p105 is as follows:
The nucleic acid sequence of p105 is as follows:
The amino acid sequence of p50 is as follows:
The amino acid sequence of KPC1 (human) is as follows:
For example, mouse (Mus musculus) KPC1 (SEQ ID No. 6) is as follows:
The mouse (Mus musculus) KPC1 (SEQ ID No. 6) is encoded by SEQ ID No. 7:
In some embodiments of the invention, there is provided a method for treating cancer comprising the step of administering a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1; a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1; or a complex comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or the agent that upregulates KPC1; or the fused protein that comprises KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, to a subject in need, thereby treating cancer.
In some embodiments of the invention, there is provided a method for treating cancer comprising the step of administering a therapeutically effective amount of functionally related variant of the KPC1 or a functionally active fragment of KPC1, a fused protein comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1 or a complex comprising the functionally related variant of the KPC1 or the functionally active fragment of KPC1, or the fused protein that comprises the functionally related variant of the KPC1 or the functionally active fragment of KPC1, to a subject in need, thereby treating cancer.
In some embodiments of the invention, there is provided a method for treating cancer comprising the step of administering a therapeutically effective amount of p50, a peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or an agent which upregulates p50; a fused protein comprising p50 or the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3, or the agent which up-regulates p50; or a complex comprising p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50, or the fused protein that comprises p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50, to a subject in need, thereby treating cancer.
In some embodiments of the invention, there is provided a method for treating cancer comprising the step of administering a therapeutically effective amount of functionally related variant of the p50 or a functionally active fragment of p50, a fused protein comprising the functionally related variant of the p50 or the functionally active fragment of p50 or a complex comprising either the functionally related variant of the p50 or the functionally active fragment of p50, the fused protein that comprises the functionally related variant of the p50 or the functionally active fragment of p50 to a subject in need, thereby treating cancer.
In some embodiments of the invention, there is provided a functionally equivalent molecule that mimics a functional activity of the KPC1 or the functionally related variant thereof, wherein the molecule is a peptidomimetic or a stapled peptide or a chemical compound.
In some embodiments of the invention, there is provided a functionally equivalent molecule that mimics a functional activity of the p50 or the functionally related variant thereof wherein the molecule is a peptidomimetic or a stapled peptide or a chemical compound.
As used in the term “protein(s) of the invention” peptide(s) of the invention”, active ingredient(s) of the invention”, “the peptide(s)”, “the protein (s)”, the “active ingredient(s)” refers inter-alia to any of the following:
KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, an agent which upregulates KPC1; a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, or a complex comprising either KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which upregulates KPC1, or a fused protein that comprises KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which upregulates KPC1; a functionally related variant of the KPC1 or a functionally active fragment of KPC1 or a fused protein comprising them, p50, a peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or an agent which upregulates p50; or a fused protein or a complex comprising them; a functionally related variant of the p50 or the functionally active fragment of p50 or a fused protein or a complex comprising them. Further included is a functionally equivalent molecule that mimics a functional activity of these proteins or peptides.
In one embodiment of the invention, this invention provides a functionally equivalent molecule that mimics the functional activity of any of the peptide or peptide variants provided in this invention. The term “functionally equivalent molecule” refers in the application to any compound such as but not restricted to peptidomimetic or stapled peptide. The functionally equivalent molecule may be obtained by retro-inverso or D-retro-enantiomer peptide technique, consisting of D-amino acids in the reversed sequence. The functionally equivalent molecule may be obtained by using amino acid derivative.
In some embodiments of the invention, there is provided a method for treating cancer comprising the step of contacting cancerous cells of the subject with a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1, a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, or a complex comprising, KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the fused protein that comprises KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, to a subject in need, thereby treating cancer.
According to some embodiments of the invention, the contacting is effected in-vivo.
According to some embodiments of the invention, the contacting is effected ex-vivo.
As used herein the term “treating cancer” refers to preventing, curing, reversing, attenuating, alleviating, minimizing or suppressing the cancer, as well as resulting in one or more of the following parameters: reduction in tumor size or burden, blocking of tumor growth, shifting the phenotype of the macrophage from M2 to M1, reduction in tumor-associated pain, long-term non-progression, induction of remission, reduction of metastasis, or increased patient survival.
As used herein the term “cancer” refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. Typically, the cancer cells are in the form of a tumor; existing locally within an animal, or circulating in the blood stream as independent cells, for example, leukemic cells.
In some embodiments of the invention, the cancer is an oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, a urogenital cancer, a gastrointestinal cancer, a central or peripheral nervous system tissue cancer, an endocrine or neuroendocrine cancer or a hematopoietic cancer.
According to some embodiments of the invention, the cancer is a glioma, a sarcoma, a carcinoma, a lymphoma, a melanoma, a fibroma, or a meningioma.
According to some embodiments of the invention, the cancer is brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary cancer, prostatic cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma tumors, multiple neuroendocrine type I and type II tumors, breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
In some embodiments of the invention, the cancer is a breast cancer, a pancreatic cancer or a lung cancer.
In some embodiment the cancer is breast cancer, bone osteosarcoma or glioblastoma.
In some embodiments of the invention, there is provided a method of preventing or reducing metastasis comprising the step of administering a therapeutically effective amount of KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1; a fused protein comprising KPC1, the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1; or a complex comprising KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the fused protein that comprises KPC1 or the peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or the agent which up-regulates KPC1, to a subject in need.
In some embodiments of the invention, there is provided a method of preventing or reducing metastasis comprising the step of administering a therapeutically effective amount of p50, a peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or an agent which upregulates p50; a fused protein comprising p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which up-regulates p50, or a complex comprising p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50 or a fused protein that comprises p50, the peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or the agent which upregulates p50.
In some embodiments there is provided a method of treating cancer by administering a therapeutically effective amount of a nucleic acid sequence that encodes to KPC1, or to a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4, or a nucleic acid that encodes to a fused protein comprising KPC1 or peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or a nucleic acid that encodes to an agent which up-regulates KPC1 or the fusion protein comprising the same. In some embodiments, the nucleic acid is as set for in SEQ ID. No. 5.
In some embodiments there is provided a method of treating cancer by administering a therapeutically effective amount of a nucleic acid sequence that encodes to p50 or to a peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3, or a nucleic acid that encodes to a fused protein comprising p50 or peptide which is at least about 70% homologous to the p50 sequence as set forth in SEQ ID No. 3 or a nucleic acid that encodes to an agent which up-regulates the processing of p105 to p50.
The method may further comprise administering a second anti-cancer therapy or a third anti-cancer therapy to the treated subject. The second and third anti-cancer therapies may be one or two or more of chemotherapy, radiotherapy, hormonal therapy, cytokine therapy, immunotherapy, targeted therapy, e.g., bortezomib, sunitinib, Herceptin, sorafenib and/or surgery. The second and third anti-cancer therapy may be administered to the subject prior to or after the KPC1 treatment or concurrent with the KPC1 treatment.
In some embodiments, the method of treatment may further comprise assessing the efficacy of the treatment by performing a PET scan on said subject or measuring the level of the relevant bio-markers.
Specifically, KPC1 SEQ ID. No. 4, or p50 SEQ ID No. 3, which form a part of the invention also refers to homologs (e.g., polypeptides), which are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 91%, at least about 93%, at least about 95%, at least about 97% or more, homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or homologous to the p50 sequence as set forth in SEQ ID No. 3 listed herein, as determined using any appropriate means, including BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters). The homolog may also refer to a deletion, insertion, or substitution variant, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof.
As used herein the term “about” refers to ±10%.
As used herein, in one embodiment, the term “amino acid derivative” refers to a group derivable from a naturally or non-naturally occurring amino acid, as described and exemplified herein. Amino acid derivatives are apparent to those of skill in the art and include, but are not limited to, ester, amino alcohol, amino aldehyde, amino lactone, and N-methyl derivatives of naturally and non-naturally occurring amino acids. In an embodiment, an amino acid derivative is provided as a substituent of a compound described herein, wherein the substituent is —NH-G(Sc)—C(0)-Q or —OC(0)G(Sc)-Q, wherein Q is —SR, —NRR or alkoxyl, R is hydrogen or alkyl, Sc is a side chain of a naturally occurring or non-naturally occurring amino acid and G is C1-C2 alkyl. In certain embodiments, G is Ci alkyl and Sc is selected from the group consisting of hydrogen, alkyl, heteroalkyl, arylalkyl and heteroarylalkyl.
As used herein, in one embodiment, the term “peptide” may be derived from a natural biological source, synthesized, or produced by recombinant technology. It may be generated in any manner, including by chemical synthesis. One or more of the amino acids may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofamesyt group, a fatty acid group, an acyl group (e.g., acetyl group), a linker for conjugation, functionalization, or other known protecting/blocking groups.
As used herein, in one embodiment, the term “peptide,” may be fragments, derivatives, analogs, or variants of the foregoing peptides, and any combination thereof. Fragments of peptides, as that term or phrase is used herein, include proteolytic fragments, as well as deletion fragments. Variants of peptides include fragments and peptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions.
Variants may occur naturally or be non-naturally occurring. Examples include fusion proteins, peptides having one or more residues chemically derivatized by reaction of a functional side group, and peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. These modifications may also include the incorporation of D-amino acids, or other non-encoded amino-acids. In one embodiment, none of the modifications should substantially interfere with the desired biological activity of the peptide, fragment thereof. In another embodiment, modifications may alter a characteristic of the peptide, fragment thereof, for instance stability or half-life, without interfering with the desired biological activity of the peptide, fragment thereof. In one embodiment, as used herein the terms “peptide” and “protein” may be used interchangeably having all the same meanings and qualities.
In one embodiment, peptide of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the peptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the peptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the peptide and the cleavable moiety and the peptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].
In one embodiment, the peptide of the present invention is retrieved in a substantially pure form.
In one embodiment, the phrase “substantially pure” refers to a purity that allows for the effective use of the protein in the applications described herein.
In one embodiment, the peptide of the present invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.
In one embodiment, production of a peptide of this invention is using recombinant DNA technology. A “recombinant” peptide, or protein refers to a peptide, or protein produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired peptide or protein.
In some embodiments, the recombinant peptides, fragments thereof or peptides are synthesized and purified; their therapeutic efficacy can be assayed either in vivo or in vitro. In one embodiment, the activities of the recombinant fragments or peptides of the present invention can be ascertained using various assays including cell viability, survival of transgenic mice, and expression of megakaryocytic and lymphoid RNA markers.
In one embodiment, a peptide of this invention comprises at least 3 amino acids. In another embodiment, a peptide comprises at least 5 amino acids. In another embodiment, a peptide comprises at least 10 amino acids. In another embodiment, a peptide comprises at least 20 amino acids. In another embodiment, a peptide comprises at least 25 amino acids. In other embodiments, a peptide comprises at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids or 300 amino acids or 350 amino acids or 400 amino acids. In one embodiment, a peptide of this invention consists essentially of at least 5 amino acids. In another embodiment, a peptide consists essentially of at least 10 amino acids. In other embodiments, a peptide consists essentially of at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids or 300 amino acids or 350 amino acids or 400 amino acids. In one embodiment, a peptide of this invention consists of at least 5 amino acids. In another embodiment, a peptide consists of at least 10 amino acids. In other embodiments, a peptide consists of at least 30 amino acids or at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acids or 300 amino acids or 350 amino acids or 400 amino acids.
As used herein, in one embodiment, the terms “peptide” and “fragment” may be used interchangeably having all the same meanings and qualities. As used herein in, in one embodiment the term “peptide” includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into bacterial cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided herein under.
Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds (—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylalanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
As used herein, in one embodiment the term “amino acid” refers to naturally occurring and synthetic α, βγ or δ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. In certain embodiments, the amino acid is in the L-configuration. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. As used herein, in one embodiment the phrase “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. “Amino acid variants” refers to amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated (e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations”, which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant”, including where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Guidance concerning which amino acid changes are likely to be phenotypically silent can also be found in Bowie et al., 1990, Science 247: 1306 1310. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. Typical conservative substitutions include but are not limited to: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids can be substituted based upon properties associated with side chains, for example, amino acids with polar side chains may be substituted, for example, Serine (S) and Threonine (T); amino acids based on the electrical charge of a side chains, for example, Arginine (R) and Histidine (H); and amino acids that have hydrophobic side chains, for example, Valine (V) and Leucine (L). As indicated, changes are typically of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically or pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Further included are constructs which include nucleic acid encoding the same the proteins of the invention.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
In some embodiments, the invention further envisages inclusion of the proteins of the invention sequence or a fused protein thereof in a complex where it is attached to proteinaceous (e.g., heterologous amino acid sequence) or non-proteinaceous moieties (e.g., PEG), each of which being capable of prolonging the half-life of the composition while in circulation.
Such a molecule is highly stable (resistant to in-vivo proteaolytic activity, probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis. Further recombinant techniques may still be used, whereby the recombinant peptide product is subjected to in-vitro modification (e.g., PEGylation as further described herein below).
The phrase “non-proteinaceous moiety” as used herein refers to a molecule not including peptide bonded amino acids that is attached to the above-described KPC1 or p50 amino acid sequence. According to some embodiments the non-proteinaceous moiety may be a polymer or a co-polymer (synthetic or natural). Non-limiting examples of the non-proteinaceous moiety of the present invention include polyethylene glycol (PEG) or derivative thereof, Polyvinyl pyrrolidone (PVP), albumin, divinyl ether and maleic anhydride copolymer (DIVEMA); polysialic acid (PSA) and/or poly(styrene comaleic anhydride) (SMA). Additionally, complexes which can protect KPC1 or p50 from the environment and thus keep its stability may be used, including, for example, liposomes or micelles containing the active ingredient of the invention or a fused protein comprising thereof are also included in the invention.
According to some embodiments of the invention, the active ingredient of the invention or the fused protein comprising the active ingredient of the invention is attached to a non-proteinaceous moiety, which may act as a sustained-release enhancing agent. Exemplary sustained-release enhancing agents include, but are not limited to hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), glyme and polyisopropylacrylamide.
Attaching the amino acid sequence component of the active ingredient of the invention or the fused protein comprising thereof of the invention to other non-amino acid agents may be by covalent linking or by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the active ingredient of the invention or the fused protein comprising thereof in liposomes or micelles to produce a complex comprising the active ingredient of the invention or the fused protein comprising the same. The association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final peptide of the invention.
In some embodiments, the PEG derivative is N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfide, carbonyldiimidazole-activated PEGs, PEG-thiol, PEG-maleimide. PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC) or PEG-orthopyridyl disulfide may be also used.
The non-proteinaceous moiety may be attached to the active ingredient of the invention amino acid sequence in any chosen position, provided that the therapeutic activity of the proteins of the invention (which may be in some embodiments KPC1 or p50) is retained.
In some embodiments, the conjugated active ingredient of the invention molecules are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC).
Molecules of this aspect of the present invention may be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation and classical solution synthesis.
Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
In instances where large amounts of the peptides of the present invention are desired, they may be produced using recombinant techniques such as described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988&, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
In some embodiments of the invention, there is provided a fused protein that comprises KPC1 or p50 as defined herein together with one or more molecule which extend the half life of KPC1 or p50 in the plasma. In some embodiments, the fused protein further comprises a linker. In some embodiments of the invention, there is provided a fused protein that comprises KPC1 or p50 as defined herein and a protein that stabilizes KPC1 or p50 as defined herein or protect it in the blood stream or at the tissue. In some embodiments the fused protein comprises KPC1 or p50 as defined herein attached to a heterologous amino acid sequence. In some embodiments, the heterologous amino acid sequence comprises an immunoglobulin amino acid sequence.
In some embodiments of the invention, there is provided a fused protein that comprises KPC1 or p50 as defined herein and IgG. The IgG may any subclasses or isotypes thereof, e.g., IgG1, IgG2, IgG3, IgG4. In some embodiments any other immunoglobulin region may be used.
In some embodiments, the term “antibody” refers to the structure that constitutes the natural biological form of an antibody. In most mammals, including humans, and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In each pair, the light and heavy chain variable regions (VL and VH) are together responsible for binding to an antigen, and the constant regions (CL, Cγ1, Cγ2, and Cγ3, particularly Cγ2, and Cγ3) are responsible for antibody effector functions. In some mammals, for example in camels and llamas, full-length antibodies may consist of only two heavy chains, each heavy chain comprising immunoglobulin domains VH, Cγ2, and Cγ3. By “immunoglobulin (Ig)” herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to VH, Cγ1, Cγ2, Cγ3, VL, CL, Fab and Fc fragments.
Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes”. There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
In some embodiments, a construct which includes a nucleic acid sequence for KMPC1 as defined herein, for example, SEQ ID. No. 5, and a nucleic acid sequence for IgG may be used in the construct. In some embodiments of the invention, the KPC1 or p50 as defined herein and the IgG are directly fused to each other.
Serum albumin can also be engaged in half-life extension through modules with the capacity to non-covalently interact with albumin. In these approaches, an albumin-binding moiety is either conjugated or genetically fused to the therapeutic protein Proteins with albumin-binding activity are known from certain bacteria. For example, streptococcal protein G contains several small albumin-binding domains (ABD) composed of roughly 50 amino acid residues (6 kDa). Fusion of an ABD to a protein results in a strongly extended half-life (see Roland E Kontermann, strategies for extended serum half-life of protein therapeutics, Current Opinion in Biotechnology 2011, 22:868-876.
Furthermore, the present invention encompasses nucleic acids encoding the fusion proteins described herein. In addition, vectors comprising these nucleic acids and cells transformed with theses vectors are encompassed by the present invention.
Briefly, the fused protein is prepared as follows: an expression construct (i.e., expression vector), which includes an isolated polynucleotide (i.e., isolated from a naturally occurring source thereof that comprises a nucleic acid sequence encoding the KPC1 or p50 as defined herein amino acid sequence fused (optionally including a linker) in frame to a nucleic acid sequence encoding the IgG amino acid sequence e.g., AB776838 (for human, NCBI database) or DQ38154 (for mouse, NCBI database), positioned under the transcriptional control of a regulatory element, such as a promoter, is introduced into host cells.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the pharmaceutical composition in a local, rather than systemic, manner, for example, via injection of the pharmaceutical composition directly into a specific tissue region of a patient.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
According to some embodiments of the invention, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (angiogenesis related disease or cancer) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, and depends on the severity of the disease, its type, the mode of administration and the like.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
Dosage amount and interval may be adjusted individually to ensure levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The invention is further related to the treatment of diseases that are associated or augmented by inflammation, bacterial translocation and or gut flora derangement, such as, for example, without being limited, chronic liver diseases and Alzheimer disease, hepatic encephalopathy, ADHD, metabolic syndrome, diabetes both type 1 and type 2, atherosclerosis or chronic fatigue syndrome, NASH, obesity, hepatic encephalopathy and potentially several immune mediated disorders among them Alopecia Areata, Lupus, Anlcylosing Spondylitis, Meniere's Disease, Antiphospholipid Syndrome, Mixed Connective Tissue Disease, Autoimmune Addison's Disease, Multiple Sclerosis, Autoimmune Hemolytic Anemia, Myasthenia Gravis, Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet's Disease, Pernicious Anemia, Bullous Pemphigoid, Polyarthritis Nodosa, Cardiomyopathy, Polychondritis, Celiac Sprue-Dermatitis, Polyglandular Syndromes, Chronic Fatigue Syndrome (CFIDS), Polymyalgia Rheumatica, Chronic Inflammatory Demyelinating, Polymyositis and Dermatomyositis, Chronic Inflammatory Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST Syndrome, Raynaud's Phenomenon, Cold Agglutinin Disease, Reiter's Syndrome, Crohn's Disease, Rheumatic Fever, Discoid Lupus, Rheumatoid Arthritis, Essential Mixed, Cryoglobulinemia Sarcoidosis, Fibromyalgia, Scleroderma, Grave's Disease, Sjogren's Syndrome, Guillain-Barre, Stiff-Man Syndrome, Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA Nephropathy, Uveitis, Insulin Dependent Diabetes (Type I), Vasculitis, Lichen Planus, and Vitiligo. The compositions described herein can be administered to a subject to treat or prevent disorders associated with an abnormal or unwanted immune response associated with cell, tissue or organ transplantation, e.g., renal, hepatic, and cardiac transplantation, e.g., graft versus host disease (GVHD), or to prevent allograft rejection.
In some embodiments of the invention, as can be seen from Examples, the inflammation is related to gastrointestinal inflammation, such as, inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn's disease (CD).
In some embodiments of the invention, there is provided a method for producing p50 comprising contacting a cell culture preparation which expresses p105 with KPC1, a peptide which is at least about 70% homologous to the KPC1 sequence as set forth in SEQ ID No. 4 or an agent which up-regulates KPC1, thereby producing p50. In some embodiments, the cells are cells that secrete the p105 into the medium.
In some embodiments of the invention, there is provided a method for screening a potential cancer therapeutic, comprising:
(i) contacting p105 with KPC1 under conditions where p105 is cleaved to p50 by KPC; (ii) contacting p105 with KPC1 and with a test compound suspected of being a cancer therapeutic under conditions where p105 is cleaved to p50 by KPC1;
(iii) measuring the amount of p105 cleaved to p50 by KPC1 in the presence and in the absence and the test compound; and
(iv) comparing the amount of p105 cleaved to p50 by KPC1 in the presence and in the absence and the test compound, wherein an increase in cleavage in the presence of the test compound is indicative of a cancer therapeutic.
In some embodiments, the test compound act synergistically with KPC1.
EXAMPLESExperimental Procedures
Materials, Plasmids, Expressed Proteins, and Cells
All materials (including plasmids and their construction, expression of proteins and their purification, and cultured cells and their manipulation), are described under Supplemental Information.
Preparation and Fractionation of Crude Reticulocyte Lysate
Reticulocytes were induced in rabbits and lysates were prepared and fractionated over DEAE cellulose to Fraction I (unabsorbed material) and Fraction II (high salt eluate) as described (Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206-8214). Fraction II (˜200 mg) was further resolved using different successive chromatographic methods as described under Supplemental Information.
In Vitro Translation
p105 or p100 were translated in vitro in the presence of L-[35S]methionine using the TNT® T7 Quick reticulocyte lysate-based coupled transcription-translation kit according to the manufacturer's instructions.
In Vitro Conjugation and Processing of p105
Ub conjugation and processing of 35S-labeled p105 were carried out in a reconstituted cell free system containing crude Fraction II as described (Kravtsova-Ivantsiv, Y., Cohen, S., and Ciechanover, A. (2009). Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor. Mol. Cell 33, 496-504). For conjugation, 1 μg of purified Kpc1-FLAG-TEV-6×HIS, Kpc1I1256A-FLAG-TEV-6×HIS, or 6×His-KPC2 were added as indicated, instead of Fraction II.
Ub Conjugates in Cells
HEK293 cells were transfected with control siRNA or siRNA against KPC1 as described herein. After 24 hr, the cells were transfected with cDNAs coding for FLAG-p105 proteins along with cDNAs coding for HA-Ub and Myc-KPC1, or with an empty vector. After additional 24 hr, the proteasome inhibitor MG132 (20 μM) was added for 3 hr, and the cells were lysed with RIPA buffer supplemented with freshly dissolved iodoacetamide and N-ethylmaleimide (5 mM each) to inhibit deubiquitinating enzymes. p105 (both free and ubiquitinated) and free p50 were immunoprecipitated with immobilized anti-FLAG. The beads were washed five times with RIPA buffer and proteins were resolved by SDS-PAGE. Free and conjugated p105 (and free p50) were visualized using anti-FLAG.
Tumorigenicity
Cell-based (soft agar) and animal (mice xenografts) tumorigenicity assays are described under Supplemental Information.
RNASeq Analysis
RNA from U87-MG xenografts was isolated using RNA purification kit and analyzed using the Illumina HiSeq 2500 analyzer. Identification and clustering of the human genes are described under Supplemental Information.
Immunohistochemistry and Statistical Analysis
The staining technique and statistical analysis of the staining data of SCCHN, breast cancer and glioblastoma were performed as described under Supplemental Information.
Supplemental Information
Extended Experimental Procedures
Materials
Materials for SDS-PAGE and Bradford reagent were from Bio-Rad. L-[35S]methionine and pre-stained MW markers were from GE Healthcare. Tissue culture sera, media, and supplements, were from Sigma or from Biological Industries [Bet HaEmek, Israel; except for the FCS used for growing Sf9 cells that was from Hyclone, and Iscove's Modified Dulbecco's Medium (IMDM) for growing HAP1 cells that was from Gibco®]. HAP1 control cells (Carette et al., 2011) and HAP1 cells knocked out for KPC1 [1 bp insertion (nucleotide is underlined and bolded) in exon 4 (NM_022064; 192-CCAGAACATTTGGACCAGTTGGCTACAGGTGGACAATGAGG-232 (SEQ ID No. 8); the insertion results in a frameshift] or KPC2 [1 bp insertion (nucleotide is underlined and bolded) in exon 2 (NM_016172; 210-GTGCTGAGT GATGCCAGGACACATCCTGGAAGAGAACATCC-250 (SEQ ID No. 9); the insertion results in a frameshift] were generated by Haplogen Genomics GmbH (Vienna, Austria), using the Crispr-CAS technology. Collagenase I was from Sigma and Dispase II from Roche. Free and immobilized mouse anti-FLAG (M2), rabbit anti-p50 Prestige Antibodies® (for immunoprecipitation and Western blot), and FLAG® peptide, were from Sigma. Mouse anti-HA (16B12) was from Covance, and rabbit anti-p50 (NLS) and its blocking peptide, mouse anti-KPC1 (267.1 for Western blot), anti-p65 (A), anti-p27 (C-19) and anti-GFAP (H-50), were from Santa Cruz. Anti-KPC1 (ab57549 for immunohistochemistry and for Western blot) was from Abcam, whereas anti-cleaved caspase 3 (D175) was from Cell Signaling. Anti-ki-67 (MIB-1) was from DAKO, and anti-actin was from Millipore. Peroxidase-conjugated (for Western blotting) and Rhodamine Red™-X-conjugated goat anti-rabbit (for immunofluorescence) secondary antibody were from Jackson ImmunoResearch Laboratories. VECTASHIELD® Mounting Medium with DAPI was from Vector Laboratories. Secondary HRP-conjugated antibody HISTOFINE® Simple Stain™, Max Po Universal Immuno Peroxidase Polymer anti-rabbit/anti-mouse, and HISTOFINE® Simple Stain™ AEC solution for immunohistochemistry, were from Nichirei Biosciences. Ubiquitin, dithiothreitol (DTT), phosphocreatine, creatine phosphokinase, adenosine 5′-triphosphate (ATP), adenosine 5′-[γ-thiotriphosphate] (ATPγ S), iodoacetamide, N-ethylmaleimide, Tris and HEPES buffers, paraformaldehyde, crystal violet, and O-nitrophenyl-beta-D-galactopyranoside (ONPG), were from Sigma. Protease inhibitors mixture and N-carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG132) were from Calbiochem. Ub aldehyde (UbA1) was from BIOMOL. Reagents for enhanced chemiluminescence (ECL) were from Pierce. TNT® T7 Quick reticulocyte lysate-based coupled transcription/translation kit and the luciferase reporter 1000 assay system were from Promega. JetPEI™ cell transfection reagent was from Polyplus. Lipofectamine® RNAiMAX and Lipofectamine® 2000 transfection reagents for siRNA and for DNA transfection, respectively, and Bac-to-Bac® baculovirus expression system, were from Invitrogene. siRNAs were synthesized by Dharmacon. shRNAs, RevertAid H Minus First Strand cDNA Synthesis Kit, Shandon Immune-Mount™, and LightShift® Chemiluminescent ElectroMobility Shift Assay (EMSA) kit, were from Thermo Scientific. TaqMan Fast Universal PCR Master Mix and TaqMan Gene Expression Assay were from Applied Biosystems. Restriction and modifying enzymes were from New England Biolabs. Oligonucleotides were synthesized by Syntezza Bioscience or by Sigma. All the chromatographic columns were purchased from GE Healthcare except for the hydroxyapatite column that was from BioRad. Low Melt Agarose and mini-PROTEAN® TBE precast gel (5%, for separation of dsDNA) were from Bio-Rad. DEAE cellulose was purchased from Whatman. Ni-NTA resin was from QIAGEN. Glioblastoma and breast tissue microarrays were from US Biomax, Inc. NucleoSpin® Kit for RNA purification was from Macherey-Nagel. All other reagents were of high analytical grade.
Plasmid Construction
cDNAs coding for human p105 and p105S927A for in vitro translation (in pT7β-6×HIS) and transient transfection in cells (in pFLAG-CMV2), were described previously (Cohen, S., Achbert-Weiner, H., and Ciechanover, A. (2004). Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing. Mol. Cell Biol. 24, 475-486; Cohen, S., Lahav-Baratz, S., and Ciechanover, A. (2006). Two distinct ubiquitin-dependent mechanisms are involved in NF-kappaB p105 proteolysis. Biochem. Biophys. Res. Commun. 345, 7-13). For in vitro translation of deleted species of p105 (p105Δ500-600, p105Δ565-660, p105Δ647-758, p105Δ752-794, p105Δ544-803), the corresponding cDNAs were generated by PCR and cloned into the pT7β-6×HIS BamHI and EcoRI restriction sites. For transient transfection of the deleted species of p105 in cells, the cDNA fragments with the deletions that were cloned initially into pT7β-6×HIS, were sub-cloned into the PstI restriction site in pFLAG-CMV2p105 or pFLAG-CMV2p105S927A. FLAG-p105K594,625,630,637,639,640,684,740R (FLAG-p105K8R; where all the lysine residues in the ankyrin repeats and in between them were substituted with arginines) in pFLAG-CMV2, was generated by site-directed mutagenesis. FLAG-p105Δ574-803 (in which all but one of the ankyrin repeats were deleted) was generated by PCR and was cloned into the pFLAG-CMV2 NotI restriction site. cDNA coding for p100 was amplified with primers flanked with HindIII and BamHI restriction sites, and was cloned into pFLAG-CMV2.
cDNAs coding for human p105K46-58R, p105Δ429-654, and p105Δ429-654;K29,K46-58R for in vitro translation (in pT7β-6×HIS), and p105Δ429-654 and p105Δ429-654;K29,K46-58R for transient transfection (in pFLAG-CMV2), were described previously (Cohen, S., Achbert-Weiner, H., and Ciechanover, A. (2004). Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing. Mol. Cell Biol. 24, 475-486; Cohen, S., Lahav-Baratz, S., and Ciechanover, A. (2006). Two distinct ubiquitin-dependent mechanisms are involved in NF-kappaB p105 proteolysis. Biochem. Biophys. Res. Commun. 345, 7-13). The cDNA coding for p105K46-58R was amplified (using pT7βp105K46-58R as a template) with primers flanked with NotI restriction site, and was cloned into pFLAG-CMV2.
cDNAs coding for HA-Ub, Myc-KPC1, and Myc-KPC1I1256A for expression in mammalian cells sub-cloned into pCAGGS (Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199), were provided by Dr. Kazuhiro Iwai.
KPC1-FLAG-TEV-6×HIS and KPC1I1256A-FLAG-TEV-6×HIS for expression in insect cells were cloned into pFastBac™ via several steps. Initially, the N-terminal segment of KPC1 was amplified with primers flanked with BsshII and EcoRI, and the C-terminal segment of KPC1 was amplified with primers flanked with EcoRI and SalI. The two fragments were sub-cloned into the appropriate restriction sites of pFastBac™. Finally, FLAG-TEV-6×HIS was introduced into pFastBac™ using the SalI restriction site.
cDNA coding for KPC2 for expression in bacterial cells was amplified with primers flanked with EcoRI and HindIII restriction sites, and was sub-cloned into pT73-6×HIS.
cDNAs coding for FLAG-IKKβ and the constitutively active FLAG-IKKβS176,180E were as described (Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997). IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 278, 860-866).
cDNA coding for NIK was as described (Senftleben, U., Cao, Y., Xiao, G., Greten, F. R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S. C., and Karin, M. (2001). Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway. Science 293, 1495-1499).
cDNA coding for 6×HIS-E6-AP was as described (Zaaroor-Regev, D., de Bie, P., Scheffner, M., Noy, T., Shemer, R., Heled, M., Stein, I., Pikarsky, E., and Ciechanover, A. (2010). Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome. Proc. Natl. Acad. Sci USA 107, 6788-6793).
cDNAs coding for Myc-KPC1, Myc-KPC1I1256A, and FLAG-p50 for generation of cells that stably express these proteins, were amplified with primers flanked with XhoI and BamHI or with XhoI, respectively, and were sub-cloned into the NSPI-CMV MCS lentiviral expression vector (Akiri, G., Cherian, M. M., Vijayakumar, S., Liu, G., Bafico, A., and Aaronson, S. A. (2009). Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene 28, 2163-2172).
cDNA coding for p65 was amplified with primers flanked with BamHI and was sub-cloned into pT7β-6×HIS BamHI restriction site.
Cultured Cells
HEK293, HeLa, U20S, MDA-MB 231 and U87-MG were grown at 37° C. in DMEM supplemented with 10% fetal calf serum and antibiotics (penicillin-streptomycin). Sf9 cells were grown in Grace's medium supplemented with 10% FCS, penicillin (100 U/ml)/streptomycin (0.1 mg/ml), and yeastolate and lactalbumin (3.332 gr/l each). HAP1 cells were grown in IMDM supplemented with 10% fetal calf serum and antibiotics (penicillin-streptomycin).
Synthesis of p105-Derived Peptides
Synthetic phosphorylated and non-phosphorylated peptides derived from the p105 IKKβ-phosphorylation site (917-DELRDSDSVCDS(P)GVETS(P)FRKLSFTES-942 (SEQ ID No. 10)) were prepared according to Fmoc-solid phase peptide synthesis strategy using HCTU (1-[Bis(dimethylamino)methylen]-5-chlorobenzotriazolium 3-oxide hexafluorophosphate, N,N,N′,N′-Tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium hexafluorophosphate) and DIEA (N-Diisopropylethylamine) as coupling reagents. The synthesis was carried out on a Rink Amide resin using automated peptides synthesizer (CSBIO). Phosophorylated Ser was coupled as Fmoc-Ser(HPO3Bzl)OH. Cleavage of the peptide was performed using a mixture of 85:5:5:2.5:2.5 of TFA:water:thioanisole:phenol:ethanedithiol for 2 hours at room temperature. The peptide was precipitated using cold ether, dissolved in 50% acetonitile in water in the presence of 0.1% TFA. Purification was carried using preparative column (Jupiter 10 micron, C18/C4 300 Å, 250×22.4 mm) and a linear gradient of 5-50% buffer containing 99.9% acetonitrile and 0.1% TFA over 30 min and with a flow rate of 15 ml/min. Fractions were analyzed by mass spectrometry using LCQ Fleet Ion Trap instrument (Thermo Scientific), and the fractions which showed over 85% purity were collected and lyophilized for use in the inhibition assay.
Fractionation of Crude Reticulocyte Lysate
Fraction II (˜200 mg) was resolved on a HiLoad™ 16/10 Q Sepharose HP column using a linear salt gradient of 0.0-0.6 M KCl. Fractions that contained p105 Ub-conjugating activity were further resolved on a HiPrep™ Heparin 16/10 FF column, and proteins were eluted using a linear salt gradient of 0.0-1.0 M NaCl. The fractions with the p105 E3 activity were subjected to hydroxyapatite chromatography. Proteins were eluted using a linear gradient of 10-700 mM KPi pH 7.0. The active fractions were applied to a Mono Q 5/50 GL column, and proteins eluted using a linear salt gradient of 0.0-0.6 M KCl. The active fractions were resolved on a gel filtration HiLoad 16/600 Superdex 200 column. Elution was carried out in a buffer containing 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 1 mM DTT. The E3 activity was eluted in a peak corresponding to an apparent native molecular size of ˜170-300 kDa, and was applied to a HiTrap™ Heparin HP column. Elution was performed with a linear salt gradient of 0.0-1.0 M NaCl. The active fractions from the last three chromatographic steps were analyzed by mass spectrometry as described below.
Mass Spectrometric Analysis
Samples were digested by trypsin, analyzed by LC-MS/MS on Orbitrap XL (Thermo), and identified by Protein Discoverer software version 1.4 against the rabbit section of the Uniprot database. The analysis was done using the Sequest search engine. The data were filtered with 1% FDR and 5 ppm accuracy.
Transient Transfection and Processing of p105 in Cells
HeLa cells were transiently transfected with the various indicated cDNAs using Lipofectamine® 2000 according to the manufacturer's protocol, and HEK293 cells were transiently transfected using the jetPEI™ reagent. 24 hr after transfection, cycloheximide (20 μg/ml) was added for the indicated times, and the cells were harvested and lysed with RIPA buffer [150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, and 1% NP-40, supplemented with freshly added protease inhibitors mixture]. Protein aliquots representing an equal number of cells were resolved via SDS-PAGE (10%) and blotted onto nitrocellulose membrane. p105 or its mutant species were visualized using anti-FLAG, and processing was expressed as the ratio between the band density of p50 and the sum of the band densities of p105 and p50, multiplied by 100%, except when indicated otherwise. Actin was used as a loading control and was detected using a specific antibody.
Stable Transfection
For stable transfection, U87-MG, U20S, and MDA-MB 231 cells were transfected with an empty vector, Myc-KPC1, Myc-KPC1I1256A, FLAG-p50, or with shRNA against p105 (clones IDs V2LHS_201580, V2LHS_201509, and V2LHS_201757) along with Myc-KPC1. Transfection was carried out using a Lentiviral transduction system, and cells were selected using puromycin (5 μg/ml).
siRNA
To silence human KPC1 and KPC2, ON-TARGETplus SMART pool siRNAs synthesized by Dharmacon was used (for KPC1—GCGCUACUAUUGGGAUGAA (SEQ ID No. 11), CAACUGGGCCUUCUCUGAA (SEQ ID No. 12), GCACAUGGCGGACCUCCUA (SEQ ID No. 13), GGUGAAGCUUCUAGGUAUA (SEQ ID No. 14); for KPC2—GCUAAUUGAACACGCAGAA (SEQ ID No. 15), GCACGUAGGUGGCGUUGUU (SEQ ID No. 16), CAGAAUGCCGCGUGCGAGU (SEQ ID No. 17), AGAGAUGAGCUGACGGAAA (SEQ ID No. 18)). Transfection of HEK293 cells with the siRNA oligonucleotides was performed using Lipofectamine® RNAiMAX according to the manufacturer's instructions. Briefly, HEK293 cells were grown to 85% confluence in a medium that was not supplemented with antibiotics. KPC1 or KPC2 siRNAs (40 nM) were added to the medium. The efficiency of gene expression suppression was monitored 48 hr after transfection by Western blot using anti-KPC1 or anti-KPC2.
Protein-Protein Interactions
For analyses of protein-protein interactions, HEK293 cells were transfected with cDNAs coding for FLAG-p105 proteins along with cDNA coding for Myc-KPC1 or with an empty vector. p105 proteins were immunoprecipitated with immobilized anti-FLAG, and following washing of the beads with RIPA buffer, the immunoprecipitated proteins were resolved by SDS-PAGE. p105, p50 and KPC1 were visualized using anti-FLAG or anti-KPC1. For interaction between endogenous proteins, HeLa cells were lysed with RIPA buffer, and p105 was immunoprecipitated with anti-p50. Following washing of the beads with RIPA buffer, the immunoprecipitated proteins were resolved by SDS-PAGE. p105, p50 and KPC1 were visualized using anti-p50 or anti-KPC1.
Protein Expression Using a Baculovirus Expression System
KPC1-FLAG-TEV-6×HIS and KPC1I1256A-FLAG-TEV-6×HIS were cloned into pFastBac™ vector as described above. Recombinant Baculovirus constructs were generated using Bac-to-Bac® expression system. To express the proteins, Sf9 cells were infected with the generated viruses. After 48 hr, cells were harvested and lysed in a buffer that contained 50 mM sodium phosphate buffer (pH 8.0), 600 mM NaCl, 10 mM imidazole, 1% NP40, and protease inhibitor cocktail (EDTA free).
Protein Expression Using a Bacterial Expression System
6×HIS-Ubc5c, and 6×HIS-KPC2 were transformed to Rosetta™ (DE3) pLysS Escherichia coli cells (Novagen). The bacteria were grown to 0.7 OD at 37° C., and protein expression was induced with IPTG (0.5 mM). After 4 hr, cells were harvested and lysed by sonication in a buffer that contained 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM 3-mercaptoethanol, and protease inhibitor cocktail (EDTA free). 6×HIS-E6-AP was expressed as described (Zaaroor-Regev, D., de Bie, P., Scheffner, M., Noy, T., Shemer, R., Heled, M., Stein, I., Pikarsky, E., and Ciechanover, A. (2010). Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome. Proc. Natl. Acad. Sci USA 107, 6788-6793).
Protein Purification
HIS-tagged proteins were purified under native conditions using Ni-NTA resin according to the manufacturer's instructions.
Colony Formation in Soft Agar
3 ml of DMEM containing 0.5% Low Melt Agarose and 10% fetal calf serum were poured into a 60 mm Petri dish. The layer was covered with 0.7×104 cells suspended in 1.5 ml DMEM that contains 0.3% Low Melt Agarose and 10% fetal calf serum, followed by addition of 2 ml DMEM containing 10% fetal calf serum. Medium was changed every 3 days. After three weeks, colonies were fixed (using 4% PFA), stained (with crystal violet; 0.05%), and counted, using the OpenCFU software for colony counting (opencfu.sourceforge.net).
Tumorigenicity
Exponentially growing U87-MG or MDA-MB 231 cells were stably transfected with an empty vector (V0) or with vectors coding for Myc-KPC1, Myc-KPC1I1256A or FLAG-p50, or Myc-KPC1 along with shRNA to silence p105. Cells were dissociated with trypsin, washed with PBS, and brought to a concentration of 50×106 cells/ml. Cell suspension (5×106/0.1 ml) was inoculated subcutaneously at the right flank of 7-weeks old Balb/C nude mice (n=7). Xenograft size was determined twice a week by externally measuring the growing tumors in two dimensions using a caliper. Tumor volume (V) was determined by the equation V=L×W2×0.5, where L is the length and W the width of the xenograft. At the end of the experiment, mice were sacrificed and xenografts were resected, weighed and fixed in formalin. Paraffin-embedded 5 μm sections were stained with antibodies as described above.
Immunohistochemistry
Formalin-fixed, paraffin-embedded, 5 m tissue sections of SCCHN, mice U87-MG xenografts, and glioblastoma and breast cancer tissue microarrays, or HEK293 cultured cells were immunostained for p50, KPC1, ki-67, cleaved caspase 3, anti-p27 and GFAP, as indicated. Immunostaining was performed as following: slides were de-paraffinized and rehydrated, and endogenous peroxidase activity was quenched (for 30 min) by 3% hydrogen peroxide in methanol. Slides were then subjected to antigen retrieval by boiling (for 20 min) in 10 mM citrate buffer, pH 6.0. Slides were incubated with 10% normal goat serum in PBS for 60 min to block nonspecific binding, and were incubated (for 20 hr at 4° C.) with the specific antibody (5 μg/ml) in blocking solution. Slides were then extensively washed with PBS, incubated with a secondary HRP-conjugated antibody for 60 min, and developed using AEC solution for 5 min according to the manufacturer's instructions. Sections were stained with hematoxylin or hematoxylin and eosin, and mounted using the Shandon Immune-Mount™.
Immunofluorescence Staining
U87-MG cells (5×105) were grown on cover slips for 2 days, washed three times with PBS, and fixed/permeabilized in paraformaldehyde (4% for 20 min at room temperature), followed by incubation in Triton X-100 (0.5% for 1 min). The cover slips were washed three times with PBS, incubated with 10% normal goat serum in PBS for 60 min (to block nonspecific binding), and were incubated (for 24 hr at 4° C.) with the specific antibody (5 μg/ml) in blocking solution. Following extensive washing with PBS, the cover slips were incubated with an appropriate secondary antibody (7 μg/ml) for 60 min, followed by three washes with PBS. They were then stained with 4′-6′ diamidino-2-phenylindole (DAPI) in mounting medium. Staining was examined using confocal microscope.
Electrophoretic Mobility Shift Assay (EMSA)
Extracts from U87-MG xenografts were isolated using tissue homogenizer in lysis buffer containing 20 mM HEPES (pH 8.0), 400 mM NaCl, 1 mM EDTA (pH 8.0), 1.5 mM MgCl2, 1 mM DTT and 0.05% NP-40. For studying DNA-protein interaction, the LightShift® Chemiluminescent EMSA kit was used according to the manufacturer's instructions. Briefly, extract (5 ag of protein) was incubated with duplex biotin end-labeled oligonucleotide representing the consensus NF-κB binding site [5′-AGTTGAGGGGACTTTCCCAGGC-biotin-3′ (SEQ ID No. 19) (bolded and underlined nucleotides denotes NF-κB binding consensus)], subjected to gel electrophoresis on a native polyacrylamide gel (5%), and transferred to a nylon membrane. The biotin end-labeled DNA was detected using the Streptavidin-Horseradish peroxidase conjugate and a chemiluminescent substrate.
Quantitative Real-Time PCR (qRT-PCR)
RNA from U87-MG xenografts was isolated using an RNA purification kit and converted to cDNA using cDNA Synthesis Kit. The qRT-PCR was carried out using TaqMan Gene Expression Assay. The assay was carried out in triplicates using TaqMan primers for VCAM1, HIC1, CDKN2C, IL-6 and TES genes. HPRT gene was used as a control.
RNASeq Analysis Mapped to the Human Genome
RNA from U87-MG xenografts was isolated using an RNA purification kit, and analyzed using Illumina HiSeq 2500. The number of reads was between 25,949,993 and 39,809,768 per sample. The reads were mapped to the human genome (GRCh37) using Tophat version 2.0.9 (Trapnell, C., Pachter, L., and Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111). Up to 3 mismatches were allowed per read, with up to 3 mismatches per segment. The-b2-sensitive parameter was set. The unmapped reads were mapped later to the mouse genome as described below. The RNASeq analysis experiment was repeated twice independently for KPC1 and V0 (in each experiment, RNA was pooled from tumors derived from different animals, and different pools were analyzed in duplicate or triplicate) and once for p50 (in duplicate independent pools). Several repeated attempts to extract RNA from the p50-expressing tumors did not yield any results, as the tumors were miniscule.
Only uniquely mapped reads were counted in the analysis, using the HTSeq-count package version 0.5.3p3 with ‘intersection-nonempty’ mode (Anders, S., Pyl, P. T., and Huber, W. (2014). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics).
The counts normalization and the differential expression analysis were done using the DESeq2 package version 1.2.8 (Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550).
Computational Methods for Analyses of RNASeq Results
Bonferroni adjusted p-values were calculated for the differentially expressed genes, and adjusted p-values<0.05 were considered as significant.
Consistently up- or down-regulated genes were in the KPC1 and p50 overexpressing tumors, and selected only those that had a log 2 fold-change compared to empty-vector control of 0.7 or higher, or −0.7 or lower, respectively. Correlation was calculated as the Pearson linear correlation coefficient.
DAVID (Dennis, G. Jr., Sherman, B. T., Hosack, D. A., Yang, J., Gao, W., Lane, H. C., and Lempicki, R. A. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4, P3) was used to perform functional enrichment analysis, using the functional annotation clustering tool and default settings on either the 534 up- or 48 down-regulated genes. Annotation clusters were described with selected (most descriptive) annotations, and top selected annotation clusters are presented in
Tumor suppressor and proto-oncogene annotations were gathered from the TSGene database (Zhao, M., Sun, J., and Zhao, Z. (2013). TSGene: a web resource for tumor suppressor genes. Nucleic Acids Res. 41, D970-976) and UniProt (UniProt Consortium. (2013). Update on activities at the Universal Protein Resource (UniProt) in 2013. Nucleic Acid Res. 41, D43-47). All gene mappings between datasets were based on Ensembl Gene IDs, and significance of enrichment was calculated using the cumulative hyper geometric probability distribution function which takes into account the total number of genes measured, the number of cancer-related genes, the number of genes significantly up- or down-regulated, and the overlap between those subsets.
Data for Integrative analysis of functional annotation clusters and known functional and physical protein-protein interactions between all consistently up- and down-regulated genes (including NFKB1/p50 and KPC1;
Statistical Analysis of Staining of Cancerous and Normal Tissues for KPC1 and p50
The study included 52 patients with Squamous Cell Carcinoma of Head and Neck (SCCHN) who were diagnosed in the Department of Otolaryngology, Head and Neck Surgery, Carmel Medical Center, Haifa, Israel. The study protocol was approved by the local Institutional Review Board. Archival paraffin-embedded pathological material and surrounding normal tissue was obtained for immunohistochemical staining of KPC1 and p50. Breast cancer tissue arrays contained 85 malignant and 20 non-malignant slices. Glioblastoma tissue array contained 192 samples of glioblastoma and 16 normal brain tissue samples. Specimens were examined by a senior pathologist (I. N.) and were scored according to the intensity of staining (0: none, 1: weak; 2: moderate, 3: strong), and localization (cytoplasm versus nucleus).
The results were evaluated for normality using the Kolmogorov-Smirnov test. Correlations between variables were performed using the Pearson's or the Spearman's coefficients of correlation, for parametric or non-parametric groups, respectively. Matched analysis was done to compare staining of the normal tissue to the malignant one.
Results
Example 1Identification of KPC1 as the p105 Ub Ligase
One of the still missing links in the Ub-mediated activation pathway of NF-κB is the identity of the ligase that ubiquitinates p105, resulting in its proteasomal processing to the p50 active subunit. To identify the ligase, rabbit reticulocyte lysate was fractionated using different chromatographic principles (
To directly test the role of KPC in p105 ubiquitination and processing, a cell free conjugation assay was established using labeled p105 as a substrate and purified KPC1 or its catalytically inactive species (mutated in the RING domain) KPC1I1256A as the ligase. The WT ligase catalyzed conjugation of p105, whereas the inactive ligase did not (FIG. 2A). It appears that KPC1 activity is specific to p105, as it scarcely modifies p100, which is highly homologous to p105 and also undergoes limited proteasomal processing, most probably by a different ligase (
To demonstrate the ability of KPC1 to modify p105 in cells, Flag-p105 was overexpressed along with HA-Ub in HEK293 cells, in which KPC1 was either silenced (
KPC1 Promotes Basal and Signal-Induced Processing of p105
To demonstrate the involvement of KPC1 in p105 processing, it's the expression thereof was silenced in cells using small interfering RNA (siRNA). As can be seen in
As noted, processing of p105 occurs also following stimulation. It was further studied whether KPC1 is capable of promoting p105 processing under such conditions as well. Therefore, the generation of p50 from p105 was tested following expression of constitutively active IKKβ (IKKβS176,180E) in the presence (endogenous) or absence (silenced) of KPC1. As presented, the stimulation increased the processing of p105 (compare
In all of the described experiments, exogenously expressed p105 was used. To demonstrate the effect on endogenous p105, the human haploid cell line HAP1 was used, in which the single allele of KPC1 or KPC2 were knocked out using the Crispr-CAS technology. Elimination of KPC1 or KPC2 [that stabilizes KPC1 (Hara, T., Kamura, T., Kotoshiba, S., Takahashi, H., Fujiwara, K., Onoyama, I., Shirakawa, M., Mizushima, N., and Nakayama, K. I. (2005). Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle. Mol. Cell Biol. 25, 9292-9303); note that removal of KPC2 results in a significant decrease in the level of KPC1—
The presented finding that KPC1 mediates processing under both basal and stimulated conditions prompted the dissection of the mechanism involved. The interaction between KPC1 and p105 was monitored under basal and stimulated conditions, and it was found that expression of constitutively active IKKβ results in increased interaction between the two as assayed by co-immunoprecipitation (
To further confirm that KPC1 interacts more efficiently with phosphorylated p105, an experiment was designed in which the binding of p105 to the ligase with a synthetic phosphorylated peptide derived from the p105 IKKβ-phosphorylation site was compared. The phosphorylated peptide inhibited ubiquitination of p105 by KPC1 to a larger extent compared with its non-phosphorylated species, both in a crude system and in a system made of purified components (
Role of KPC2 in KPC1-Mediated p105 Ubiquitination and Processing
Further, the role of KPC2, the partner of KPC1 in the heterodimeric ligase complex, in p105 modification and processing was studied. It was noted that its addition to a reconstituted cell free system significantly decreases the ubiquitination of p105 by KPC1 (
Importantly, in correlation with the suppressive effect of KPC2 on KPC1-mediated ubiquitination of p105, silencing of KPC2 increased the formation of p50 (
Identification of the Ub Anchoring Sites on p105 Modified by KPC1
It has already been shown that multiple lysines in the C-terminal segment of p105 are required for its ubiquitination and processing (Cohen, S., Achbert-Weiner, H., and Ciechanover, A. (2004). Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing. Mol. Cell Biol. 24, 475-486; Kravtsova-Ivantsiv, Y., Cohen, S., and Ciechanover, A. (2009). Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor. Mol. Cell 33, 496-504) in crude extracts. It was therefore tested whether this was true also for KPC1. Progressive removal of all lysine residues from the C-terminal segment (
The C-Terminal ARs of p105 are Necessary for its Interaction with KPC1 and for its Subsequent Ubiquitination and Processing
p105 harbors several domains: REL Homology Domain (RHD), Nuclear Localization Signal (NLS), and a Glycine Rich Repeat (GRR) in its N-terminal segment, and ARs, Death Domain (DD) and a PEST (Proline, Glutamate, Serine and Threonine) sequence in the C-terminal segment (
Similar results were obtained in experiments carried out in cells. Overexpression of KPC1 increased the ubiquitination of WT p105, but much less so of p105 that lacks all its ARs (
To rule out the possibility that the decrease in ubiquitination of p105 that lacks all its ARs is due to removal of the eight lysine residues in the repeats, a mutant p105 was generated in which all those lysines were substituted by arginines. The ubiquitination of the K to R mutant as well as its interaction with KPC1, were similar to that of WT p105 (
Last, it was studied whether the ARs-dependent ubiquitination increases the processing of p105. As can be seen in
It appears that the ARs are also involved in signal-induced processing of p105, as their removal significantly decreased IKKβ-mediated generation of p50 (
Overexpression of KPC1 or p50 Suppresses Tumor Growth
Since NF-κB dimers are known to affect cell survival, proliferation and tumor progression, it was interesting to study the outcome of KPC1 on cell growth. Initially, the influence of overexpressed KPC1 on anchorage-independent growth in MB-MDA 231, U20S and U87-MG cells was monitored and it was found to inhibit colony formation by 36%, 32% and 52%, respectively, compared to controls (
These observations prompted the study of the effect of KPC1 in a tumor model in mice. Xenografts stably overexpressing V0, KPC1, KPC1I1256A or p50 were generated. Both the growth rate and weights of tumors expressing KPC1 and p50 were significantly smaller compared to those that harbor V0 or KPC1I1256A (
Of note is that all the effects on tumor growth (reduction in colony formation, tumor growth rate, and weight) were more prominent in p50-expressing tumors than in their KPC1-overexpressing counterparts. This could be explained since p50 is the product of KPC1 activity, and its direct expression has a stronger effect.
The functional linkage between KPC1 and p50 can also be observed in staining of specific proliferation and differentiation markers in the mice-derived tumors. The overexpression of KPC1, but not of KPC1I1256A, results in increased appearance of nuclear NF-κB (
KPC1 Regulates Expression of a Subset of p50 Target Genes
The profile of gene expression in the tumors using RNASeq analysis of transcripts mapped to the human genome was analyzed (Table S1 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60530). The altered gene expression patterns revealed a strong similarity between overexpression of p50 and KPC1 in U87-MG xenografts (correlation of 0.51, p-value<10-300;
Finally, an integration of functional annotation enrichment and protein-protein interactions for the differentially regulated genes was performed, revealing a dense network of up-regulated genes revolved around a few down-regulated ones, such as interleukin-6 (IL-6), interleukin-6 receptor (IL-6R), and vascular endothelial growth factor A (VEGFA) (
Taken together, the findings strongly suggest a model of KPC1/p50 driven glioblastoma tumor growth inhibition, that centers around down-regulated high mobility group AT-hook 2 (HMGA2), lin-28 homolog A (LIN28), IL-6/IL-6R, and VEGFA, and up regulated tumor suppressors, which in combination control the tumor-microenvironment as well as glioblastoma stem cell maintenance.
Example 8Correlation Between Expression of KPC1 and p50 in Human Tumoral and Normal Tissues
Finally, the relationship between KPC1 and p50 in human tumors and normal tissues was examined.
Immunohistochemical staining of the two proteins (the two antibodies were shown to be specific; see
To test the hypothesis that loss of KPC1 and nuclear p50 can be involved in the pathogenesis of malignant transformation, the staining of the two proteins in SCCHN, breast cancer, and glioblastoma were analyzed, and compared it to their staining in the normal parallel tissue. A strong decrease in tumor samples stained for nuclear p50 was observed compared to the healthy tissue (
Supplemental Information
Additional information can be found from the results demonstrated in
Discussion
The vast majority of substrates of the Ub proteasome system are completely degraded. One intriguing and exceptional case is that of the p105 precursor of NF-κB that can be either completely degraded or processed in a limited manner to yield the p50 active subunit of the transcription factor. The “decision-making” mechanism resulting in one of the two distinct processes has remained largely elusive. The □TrCP Ub ligase has been identified as the tagging enzyme involved in complete degradation of p105, whereas the ligase involved in processing has remained unknown. The KPC complex has been identified as the putative p105-processing ligase (
Now that the two E3s involved in degradation and processing of p105 have been identified, it is still not known why ubiquitination by one enzyme results in a completely different fate of p105 than ubiquitination by the other, and what determines the timing of the two reactions. It is possible that the two ligases catalyze the formation of chains that differ in their anchoring sites, length and/or internal linkages. These in turn affect the recognition and mechanism of action of the 26S proteasome. As for timing, it can be that different physiological conditions and/or the degree of saturation of the ARs with bound p50s are involved in the “decision-making” process of whether the molecule will be processed or destroyed completely.
Studying the biological implications of manipulating KPC1 revealed that it suppresses anchorage-independent growth in a manner that is dependent on its ligase activity and the presence of p105. A corollary strong tumor suppressive effect was demonstrated in xenografts of human tumors (
An important question relates to the transcriptional mechanism by which KPC1 and p50 exert their tumor suppressive effect. An obvious assumption is that the stoichiometric excess of p50 generated by KPC1 would generate mostly p50⋅p50 homodimers rather than the ‘canonical’ tumorigenic p50⋅p65 heterodimers. In line with this finding is also the observation that p65 level is decreased in KPC1—as well as in p50-overexpressing xenografts (
A strong correlation between the expression of KPC1 and that of p50 in human tumors has been found (
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
REFERENCES
- Barre, B., Coqueret, O., and Perkins, N. D. (2010). Regulation of activity and function of the p52 NF-kappaB subunit following DNA damage. Cell Cycle 9, 4795-4804.
- Barre, B., and Perkins, N. D. (2007). A cell cycle regulatory network controlling NF-kappaB subunit activity and function. EMBO J. 26, 4841-4855.
- Ben-Neriah, Y., and Karin, M. (2011). Inflammation meets cancer, with NF-kappaB as the matchmaker. Nat. Immunol. 12, 715-723.
- Betts, J. C., and Nabel, G. J. (1996). Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences. Mol. Cell Biol. 16, 6363-6371.
- Bonavia, R., Inda, M. M., Cavenee, W. K., and Furnari, F. B. (2011). Heterogeneity maintenance in glioblastoma: a social network. Cancer Res. 71, 4055-4060.
- Cohen, S., Achbert-Weiner, H., and Ciechanover, A. (2004). Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing. Mol. Cell Biol. 24, 475-486.
- Deng, W. G., and Wu, K. K. (2003). Regulation of inducible nitric oxide synthase expression by p300 and p50 acetylation. J. Immunol. 171, 6581-6588.
- DiDonato, J. A., Mercurio, F., and Karin, M. (2012). NF-kappaB and the link between inflammation and cancer. Immunol. Rev. 246, 379-400.
- Fan, C. M., and Maniatis, T. (1991). Generation of p50 subunit of NF-kappa B by processing of p105 through an ATP-dependent pathway. Nature 354, 395-398.
- Fujita, T., Nolan, G. P., Liou, H. C., Scott, M. L., and Baltimore, D. (1993). The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers. Genes Dev. 7, 1354-1363.
- Gao, J., Chen, T., Liu, J., Liu, W., Hu, G., Guo, X., Yin, B., Gong, Y., Zhao, J., Qiang, B., et al. (2009). Loss of NECL1, a novel tumor suppressor, can be restored in glioma by HDAC inhibitor-Trichostatin A through Sp1 binding site. Glia 57, 989-999.
- Hara, T., Kamura, T., Kotoshiba, S., Takahashi, H., Fujiwara, K., Onoyama, I., Shirakawa, M., Mizushima, N., and Nakayama, K. I. (2005). Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle. Mol. Cell Biol. 25, 9292-9303.
- Heddleston, J. M., Li, Z., McLendon, R. E., Hjelmeland, A. B., and Rich, J. N. (2009). The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle 8, 3274-3284.
- Heissmeyer, V., Krappmann, D., Hatada, E. N., and Scheidereit, C. (2001). Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha. Mol. Cell Biol. 21, 1024-1035.
- Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206-8214.
- Ivanov, V. N., Lee, R. K., Podack, E. R., and Malek, T. R. (1997). Regulation of Fas-dependent activation-induced T cell apoptosis by cAMP signaling: a potential role for transcription factor NF-kappa B. Oncogene 14, 2455-2464.
- Kamura, T., Hara, T., Matsumoto, M., Ishida, N., Okumura, F., Hatakeyama, S., Yoshida, M., Nakayama, K., and Nakayama, K. I. (2004). Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase. Nat. Cell Biol. 6, 1229-1235.
- Kravtsova-Ivantsiv, Y., Cohen, S., and Ciechanover, A. (2009). Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor. Mol. Cell 33, 496-504.
- Lin, L., DeMartino, G. N., and Greene, W. C. (1998). Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome. Cell 92, 819-828.
- Lin, L., and Ghosh, S. (1996). A glycine-rich region in NF-kappaB p105 functions as a processing signal for the generation of the p50 subunit. Mol. Cell Biol. 16, 2248-2254.
- MacKichan, M. L., Logeat, F., and Israel, A. (1996). Phosphorylation of p105 PEST sequence via a redox-insensitive pathway up-regulates processing of p50 NF-kappaB. J. Biol. Chem. 271, 6084-6091.
- May, M. J., and Ghosh, S. (1997). Rel/NF-kappa B and I kappa B proteins: an overview. Semin. Cancer Biol. 8, 63-73.
- Orian, A., Gonen, H., Bercovich, B., Fajerman, I., Eytan, E., Israel, A., Mercurio, F., Iwai, K., Schwartz, A. L., and Ciechanover, A. (2000). SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase. EMBO J. 19, 2580-2591.
- Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994). The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kappa B. Cell 78, 773-785.
- Perkins, N. D. (2012). The diverse and complex roles of NF-kappaB subunits in cancer. Nat. Rev. Cancer 12, 121-132.
- Perrella, M. A., Pellacani, A., Wiesel, P., Chin, M. T., Foster, L. C., Ibanez, M., Hsieh, C. M., Reeves, R., Yet, S. F., and Lee, M. E. (1999). High mobility group-I(Y) protein facilitates nuclear factor-kappaB binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer. J. Biol. Chem. 274, 9045-9052.
- Pikarsky, E., and Ben-Neriah, Y. (2006). NF-kappaB inhibition: a double-edged sword in cancer? Eur. J. Cancer 42, 779-784.
- Rahman, M. M., and McFadden, G. (2011). Modulation of NF-kappaB signalling by microbial pathogens. Nat. Rev. Microbiol. 9, 291-306.
- Salmeron, A., Janzen, J., Soneji, Y., Bump, N., Kamens, J., Allen, H., and Ley, S. C. (2001). Direct phosphorylation of NF-kappaB1 p105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis. J. Biol. Chem. 276, 22215-22222.
- Siggers, T., Chang, A. B., Teixeira, A., Wong, D., Williams, K. J., Ahmed, B., Ragoussis, J., Udalova, I. A., Smale, S. T., and Bulyk, M. L. (2012). Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-kappaB family DNA binding. Nat. Immunol. 13, 95-102.
- Voce, D. J., Schmitt, A. M., Uppal, A., McNerney, M. E., Bernal, G. M., Cahill, K. E., Wahlstrom, J. S., Nassiri, A., Yu, X., Crawley, C. D., et al. (2014). Nfkb1 is a haploinsufficient DNA damage-specific tumor suppressor. Oncogene.
- Zaaroor-Regev, D., de Bie, P., Scheffner, M., Noy, T., Shemer, R., Heled, M., Stein, I., Pikarsky, E., and Ciechanover, A. (2010). Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome. Proc. Natl. Acad. Sci USA 107, 6788-6793.
Claims
1. A method of treating cancer in a subject comprising the step of administering to said subject a therapeutically effective amount of:
- (b) an expression vector comprising a nucleic acid that encodes a fused protein comprising the KPC1 sequence as set forth in SEQ ID NO: 4,
- wherein said method comprises administering the expression vector as described in (b).
2. The method of claim 1, wherein said expression vector is comprised within a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
3. The method of claim 1, wherein the cancer is breast cancer, bone osteosarcoma, or glioblastoma.
4. A method of treating cancer in a subject comprising the step of administering to said subject a therapeutically effective amount of:
- (b) an expression vector comprising a nucleic acid that encodes a fused protein comprising the KPC1 sequence as set forth in SEQ ID NO: 4,
- wherein said fused protein comprises an immunoglobulin, an antibody, an albumin, an albumin-binding moiety, or a combination thereof.
5. The method of claim 4, wherein said immunoglobulin comprises IgG.
6. The method of claim 4, wherein said expression vector is comprised within a pharmacuetical composition comprising a pharmaceutically acceptable carrier.
7. The method of claim 4, wherein the cancer is breast cancer, bone osteosarcoma, or glioblastoma.
| 5916803 | June 29, 1999 | Sedlacek |
| 6429200 | August 6, 2002 | Monahan |
| 20040005569 | January 8, 2004 | Baker et al. |
| 20090215709 | August 27, 2009 | Van Criekinge |
| 20140079770 | March 20, 2014 | Kusumoto |
| WO-0134206 | May 2001 | WO |
| WO-02057424 | July 2002 | WO |
| WO2003100064 | December 2003 | WO |
| WO2008134752 | November 2008 | WO |
| WO-2011026641 | March 2011 | WO |
| WO-2014039012 | March 2014 | WO |
| WO-2015093557 | June 2015 | WO |
- Machine Translation of WO2015/093557, Jun. 5, 2020. (Year: 2020).
- Heitz et al, British Journal of Pharmacology, 2009, vol. 157, pp. 195-206 (Year: 2009).
- Kauffman et al (Trends in Biochemical Sciences, 2015, vol. 40, pp. 749-764) (Year: 2015).
- Wold and Toth, Author Manuscript, Current Gene Therapy, Jul. 20, 2015, 26 pages (Year: 2015).
- Appaiahgari and Vrati (Expert Opinion in Biological Therapy, 2015, vol. 15, pp. 337-351) (Year: 2015).
- Nayerssadat et al (Advances in Biomedical Research, 2012, vol. 1, No. 27, 22 pages) (Year: 2012).
- Kigel et al (PLoS One, 2008, vol. 3,e3287, 14 pages). (Year: 2008).
- Trubetskoy et al, Bioconjugate Chemistry, 1992, vol. 3, pp. 323-327 (Year: 1992).
- The abstract of Chen et al, Hyaluronan, Proceedings of the International Cellucon Conference, 2002, vol. 1, pp. 305-311 (Year: 2002).
- Mumper et al, Pharmaceutical Research, 1996, vol. 13, pp. 701-709 (Year: 1996).
- Murata et al, Bioorganic and Medicinal Chemistry Letters, 2003, vol. 13, pp. 3967-3970 (Year: 2003).
- Hayes et al, Biochimica et Biophysica Acata, 2006, vol. 1758, No. 4, pp. 429-442 (Year: 2006).
- Pitard et al, PNAS, 1997, vol. 94, pp. 14412-14417 (Year: 1997).
- Mayurkumar, International Journal of Pharmaceutics, 2013, vol. 453, pp. 400-407 (Year: 2013).
- Xiaopin, International Journal of Nanomedicine, 2012, vol. 7, pp. 4961-4972 (Year: 2012).
- Xu et al, Biomaterials, 2008, vol. 29, pp. 3023-3033 (Year: 2008).
- Huang et al, Journal of Pharmaceutical Sciences, 2002, vol. 91, pp. 1371-1381 (Year: 2002).
- Hayes et al., Biochmica et Biophysica Acta, 2006, vol. 1758, pp. 429-442 (Year: 2006).
- Kamura et al., “Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27Kip1 at G1 phase” Nature Cell Biology, Dec. 2004, vol. 6, No. 12, pp. 1229-1239.
- Saccani et al. “p50 Nuclear Factor-KB Overexpression in Tumor-Associated Macrophages Inhibits M1 Inflammatory Responses and Antitumor Resistance”, Dec. 1, 2006, The Journal Cancer Research, vol. 66 No. 23, pp. 11432-11440.
- Kravtsova-Ivantsiv et al, “The ubiquitin-proteasome system and activation of NF-κB: involvement of the ubiquitin ligase KPC1 in p105 processing and tumor suppression”, Molecular & Cellular Oncology, May 26, 2015, pp. 1-4.
- Kravtsova-Ivantsiv et al. “KPC1-Mediated Ubiquitination and Proteasomal Processing of NF-κB1 p105 to p50 Restricts Tumor Growth” Cell, Apr. 9, 2015, vol. 161, No. 2, pp. 333-347.
- Czajkowsky et al. Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 2012;4(10):1015-1028.
- Linderholm et al. Immunoglobulin FC-Fusion Proteins. BioProcess International Nov. 2014. https://bioprocessintl.com/manufacturing/monoclonal-antibodies/immunoglobulin-fc-fusion-proteins-part-2-therapeutic-uses-clinical-development.
- Dufresne & Duval. Genetic sequences: how are they patented? Nat Biotechnol. 2004;22(2):231-232.
- Reeck et al. “Homology” in proteins and nucleic acids: a terminology muddle and a way out of it. Cell. 1987;50(5):667.
- Office Action of European Patent Application No. EP16739877.5, dated Feb. 24, 2020.
- Akiri et al. (2009). Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene, 28(21), 2163-2172.
- Anders et al. (2015). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics (Oxford, England), 31(2), 166-169.
- Barré et al. (2007). A cell cycle regulatory network controlling NF-kappaB subunit activity and function. The EMBO journal, 26(23), 4841-4855.
- Barré et al. (2010). Regulation of activity and function of the p52 NF-κB subunit following DNA damage. Cell cycle (Georgetown, Tex.), 9(24), 4795-4804.
- Ben-Neriah, Y., & Karin, M. (2011). Inflammation meets cancer, with NF-κB as the matchmaker. Nature immunology, 12(8), 715-723.
- Betts, J. C., & Nabel, G. J. (1996). Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences. Molecular and cellular biology, 16(11), 6363-6371.
- Bitter et al. (1987). Expression and secretion vectors for yeast. Methods in enzymology, 153, 516-544.
- Bonavia et al. (2011). Heterogeneity maintenance in glioblastoma: a social network. Cancer research, 71(12), 4055-4060.0.
- Booth et al. (1988). The use of a ‘universal’ yeast expression vector to produce an antigenic protein of Mycobacterium leprae. Immunology letters, 19(1), 65-69.
- Bowie et al. (1990). Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science (New York, N.Y.), 247(4948), 1306-1310.
- Broglie et al. (1984). Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science (New York, N.Y.), 224(4651), 838-843.
- Cohen et al. (2004). Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing. Mol. Cell Biol. 24, 475-486.
- Coruzzi et al. (1984). Tissue-specific and light-regulated expression of a pea nuclear gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The EMBO journal, 3(8), 1671-1679.
- Dennis et al. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome biology, 4(5), P3.
- Deng and Wu (2003). Regulation of inducible nitric oxide synthase expression by p300 and p50 acetylation. J. Immunol. 171, 6581-6588.
- DiDonato et al. (2012). NF-kappaB and the link between inflammation and cancer. Immunol. Rev. 246, 379-400.
- Fan and Maniatis (1991). Generation of p50 subunit of NF-kappa B by processing of 0105 through an ATP-dependent pathway. Nature 354, 395-398.
- Fujita et al. (1993). The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers. Genes Dev. 7, 1354-1363.
- Gao et al. (2009). Loss of NECL1, a novel tumor suppressor, can be restored in glioma by HDAC inhibitor-Trichostatin A through Sp1 binding site. Glia 57, 989-999.
- Gardella et al. (1990). Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein. The Journal of biological chemistry, 265(26), 15854-15859.
- Gurley et al. (1986). Upstream sequences required for efficient expression of a soybean heat shock gene. Molecular and cellular biology, 6(2), 559-565.
- Hara et al. (2005). Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle. Mol. Cell Biol. 25, 9292-9303.
- Heddleston et al. (2009). The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle 8, 3274-3284.
- Heissmeyer et al. (2001). Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha. Mol. Cell Biol. 21, 1024-1035.
- Hershko et al. (1983). Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206-8214.
- Ivanov et al. (1997). Regulation of Fas-dependent activation-induced T cell apoptosis by cAMP signaling: a potential role for transcription factor NF-kappa B. Oncogene 14, 2455-2464.
- Kontermann (2011). Strategies for extended serum half-life of protein therapeutics. Current opinion in biotechnology, 22(6), 868-876.
- Kravtsova-Ivantsiv et al. (2009). Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-kappaB precursor. Mol. Cell 33, 496-504.
- Lin and Ghosh (1996). A glycine-rich region in NF-kappaB p105 functions as a processing signal for the generation of the p50 subunit. Mol. Cell Biol. 16, 2248-2254.
- Lin et al. (1998). Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome. Cell 92, 819-828.
- Mackichan et al. (1996). Phosphorylation of p105 PEST sequence via a redoxinsensitive pathway up-regulates processing of p50 NF-kappaB. J. Biol. Chem. 271, 6084-6091.
- May and Ghosh. (1997). Rel/NF-kappa B and I kappa B proteins: an overview. Semin. Cancer Biol. 8, 63-73.
- Mercer et al. (2012). RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection. Cell reports, 2(4), 1036-1047.
- Mercurio et al. (1997). IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 278, 860-866.
- Orian et al. (2000). SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase. EMBO J. 19, 2580-2591.
- Palombella et al. (1994). The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kappa B. Cell 78, 773-785.
- Perkins (2012). The diverse and complex roles of NF-kappaB subunits in cancer. Nat. Rev. Cancer 12, 121-132.
- Perrella et al. (1999). High mobility group-I(Y) protein facilitates nuclear factor-kappaB binding and transactivation of the inducible nitric-oxide synthase promoter/enhancer. J. Biol. Chem. 274, 9045-9052.
- Pikarsky and Ben-Neriah. (2006). NF-kappaB inhibition: a double-edged sword in cancer? Eur. J. Cancer 42, 779-784.
- Rahman and Mcfadden (2011). Modulation of NF-kappaB signalling by microbial pathogens. Nat. Rev. Microbiol. 9, 291-306.
- Salmeron et al. (2001). Direct phosphorylation of NF-kappaB1 0105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis. J. Biol. Chem. 276, 22215-22222.
- Senftleben et al. (2001). Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway. Science 293, 1495-1499).
- Siggers et al. (2012). Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-kappaB family DNA binding. Nat. Immunol. 13, 95-102.
- Studier et al. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in enzymology, 185, 60-89.
- Szklarczyk et al. (2011). The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 39, D561-568).
- Takamatsu et al. (1987). Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. The EMBO journal, 6(2), 307-311.
- Trapnell et al. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111.
- Voce et al. (2015). Nfkb1 is a haploinsufficient DNA damage-specific tumor suppressor. Oncogene, 34(21), 2807-2813.
- Weissbach & Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463.
- Zaaroor-Regev et al. (2010). Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome. Proc. Natl. Acad. Sci U S A 107, 6788-6793.
- Zhao et al. (2013). TSGene: a web resource for tumor suppressor genes. Nucleic Acids Res. 41, D970-976.
Type: Grant
Filed: Jan 5, 2016
Date of Patent: Sep 27, 2022
Patent Publication Number: 20180140667
Assignee: TECHNION RESEARCH & DEVELOPMENT FOUNDATION LIMITED (Haifa)
Inventors: Aaron Ciechanover (Caesarea), Yelena Kravtsova (Haifa), Inna Shomer (Herzilya), Victoria Cohen (Haifa)
Primary Examiner: Karen A. Canella
Application Number: 15/544,523
International Classification: A61K 38/17 (20060101); C07K 19/00 (20060101); A61P 35/00 (20060101); C12P 21/02 (20060101);
